Apparent Dissociation Rate Constants Research Articles

GS4071 Is a Slow-Binding Inhibitor of Influenza Neuraminidase from Both A and B Strains

The kinetics of inhibition of purified influenza neuraminidases from A/Tokyo/3/67 and B/Memphis/3/89 influenza viruses by (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (GS4071) were investigated. Progress curve experiments established that GS4071 is a time dependent inhibitor of both A and B strains of influenza neuraminidase. The apparent association and dissociation rate constants, as well as the overall Kivalues, were only modestly different for the two neuraminidase strains. The time dependent inhibition phenomenon, often referred to as slow-binding inhibition, appears to be a consequence of the very slow rate of dissociation of the compound from influenza neuraminidase.

The Mechanism of Binding of Low-Molecular-Weight Active Site Inhibitors to Human α-Thrombin

The thrombin inhibitors argatroban, efegatran, NAPAP, CH 1091, CH 248, inogatran and melagatran have been characterised with respect to their mechanism of binding to human alpha-thrombin. Stopped-flow spectrophotometry was used to follow thrombin-catalysed hydrolysis of the chromogenic substrate S-2238 in the presence of inhibitors. The rate of onset or decay of inhibition was evaluated using progress curve analysis. It was possible to obtain apparent association and dissociation rate constants from the dependence of the rates on the inhibitor concentrations. Inhibition constants calculated from the association and dissociation rate constants were in good agreement with those calculated from steady-state rates. The binding of 6 inhibitors was also monitored directly using stopped-flow spectrofluorimetry when two kinetic components were found with all inhibitors. The faster component accounted for the largest part of the change in the intrinsic fluorescence of thrombin induced by inhibitor binding and was dependent on the inhibitor concentration. The slower component was independent of the concentration of the inhibitor. The concentration dependence of the faster component was linear with the compounds argatroban, NAPAP, CH 1091 and melagatran and hyperbolic with the compounds CH 248 and inogatran. The values of the apparent second-order rate constants at pH 7.4 and 37 degrees C range from slow to rapid binding in the interval 16-78 x 10(6) M-1 s-1, which is somewhat higher than 1-34 x 10(6)M-1 s-1 obtained from progress curve analysis of the onset of inhibition. The present results support a mechanism that includes rearrangement of a weak initial thrombin-inhibitor complex towards a tighter complex. Moreover, at least one additional step is required in the mechanism. In this model, the rate-limiting step for the binding of the inhibitor at concentrations in the nanomolar range depends on the primary interaction between the inhibitor and native thrombin.

Na+/K+ pump inhibition and positive inotropic effect of digitoxigenin and some C-22-substituted derivatives in sheep cardiac preparations

Affinity labeling might be used to localize the binding site(s) of the lactone ring of cardioactive steroids on the Na+/K+-ATPase. The aim of the experiments described below was to identify C-22-substituted derivatives of digitoxigenin suitable for this purpose. The positive inotropic effect of digitoxigenin, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin, and 22-hydroxy-digitoxigenin was studied in sheep cardiac Purkinje fibres. In addition, the inhibition of the Na+/K+ pump by these drugs was investigated by means of simultaneous measurements of membrane current and intracellular Na+ concentration in voltage-clamped Purkinje fibres and by means of whole-cell recording in isolated sheep Purkinje cells. The experiments were performed at 5.4 mM K+ and 30 to 33 degrees C. All compounds exerted a reversible positive inotropic effect. The concentrations required for the half maximal effect (EC50 value) amounted to approximately 5 x 10(-7) M digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The EC50 values for 22-benzoyloxy-digitoxigenin and 22-allyl-digitoxigenin were estimated to be 1.3 x 10(-6) M and 1.1 x 10(-5) M, respectively. From measurements on voltage-clamped Purkinje fibres the concentrations required for half maximal Na+/K+ pump inhibition (K'D value) were calculated to be approximately 10(-6) M for digitoxigenin, 22-acetoxy-digitoxigenin or 22-hydroxy-digitoxigenin. The K'D value for 22-benzoyloxy-digitoxigenin was 10 times larger. The K'D value for 22-allyl-digitoxigenin was even larger and amounted to approximately 4 x 10(-5) M. The K'D values of the drugs derived from whole-cell recording on single Purkinje cells tended to be smaller by a factor 2 to 8. Measurements of drug binding and unbinding revealed that the apparent association rate constant of 22-benzoyloxy-digitoxigenin (approximately 9 x 10(2) s(-1) M[-1]) was smaller than the association rate constant of digitoxigenin (approximately 2 x 10(4) s(-1) M[-1]), whereas the apparent dissociation rate constants of both compounds were similar (approximately 4 x 10(-3) s[-1]). Compared to digitoxigenin 22-allyl-digitoxigenin displayed a lower association rate constant (approximately 3 x 10(3) s(-1) M[-1]) and a larger dissociation rate constant (approximately 8 x 10(-2) M[-1]). The structure-activity relationships of the drugs are discussed. We conclude that esters derived from 22-hydroxy-digitoxigenin might be suitable to localize the binding site(s) of the lactone moiety on the Na+/K+ pump by affinity labeling.

The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential

The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.

Alkaline denaturation and partial refolding of pepsin investigated with DAPI as an extrinsic probe

The binding parameters of DAPI to porcine stomach pepsin have been described in the previous article in this issue (A. Mazzini et al.). Here we exploit the differences in the spectroscopic (fluorescence and circular dichroism) properties of DAPI bound to either native or alkali denatured pepsin. We follow the kinetics of pepsin denaturation around neutrality (pH range 6.8–7.4), at several phosphate buffer ionic strengths (range 0.02–0.25). The dependence of the apparent dissociation rate constant on pH clearly shows that the rate limiting step follows the dissociation of about three acidic protein residues. The accelerating effect by ionic strength we observed can be accounted for by a simple treatment based on both transition state theory and Debye-Hueckel's limiting law. Futhermore, when a solution of pepsin, rapidly denatured at pH 7, is reacidified to a pH between 4.5 and 5.5, a substantial recovery of protein secondary structure, with no enzymatic activity, is observed, judging by the far UV circular dichroism of the protein. This process of partial refolding can easily be followed using DAPI as an extrinsic reporter group, able to monitor the kinetics of formation and decay of a highly fluorescent intermediate. This process becomes faster at a lower pH, at least in the limited range investigated (pH 4.5–5.5), in which the refolded protein does not aggregate, but, in contrast to unfolding, is almost independent in ionic strength.

Block of human cardiac Kv1.5 channels by loratadine: voltage-, time- and use-dependent block at concentrations above therapeutic levels

The aim of this study was to analyze the effects of loratadine on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line. Currents were studied using the whole-cell configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding hKv1.5 channels. Loratadine inhibited in a concentration-dependent manner the hKv1.5 current, the apparent affinity being 1.2 +/- 0.2 microM. The blockade increased steeply between -30 and 0 mV which corresponded with the voltage range for channel opening, thus suggesting that the drug binds preferentially to the open state of the channel. The apparent association and dissociation rate constants were (3.6 +/- 0.5) x 10(6).M-1.s-1 and 3.7 +/- 1.6.s-1, respectively. Loratadine, 1 microM, increased the time constant of deactivation of tail currents elicited on return to -40 mV after 500 ms depolarizing pulses to +60 mV from 36.2 +/- 3.4 to 64.9 +/- 3.6 ms (n = 6, P < 0.01), thus inducing a 'crossover' phenomenon. Application of trains of pulses at 1 Hz lead to a progressive increase in the blockade reaching a final value of 48.6 +/- 4.3%. Recovery from loratadine-induced block at -80 mV exhibited a time constant of 743.0 +/- 78.0 ms. Finally, the results of a mathematical stimulation of the effects of loratadine, based on an open-channel block model, reproduced fairly well the main effects of the drug. The present results demonstrated that loratadine blocked hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner but only at concentrations much higher than therapeutic plasma levels in man.

Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor: A comparison of immobilisation strategies

The interaction between the humanised A33 monoclonal antibody and the corresponding F(ab)2′ or Fab′ fragments with the colonic epithelial A33 antigen, purified by micropreparative HPLC from membrane extracts of the colonic carcinoma cell line LIM 1215, has been studied with the BIAcore 2000 biosensor using surface plasmon resonance detection. The surface orientation of immobilised antibody and the Fab′ fragment onto the biosensor surface was controlled using alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared with the conventional N-hydroxysuccinimide (NHS)/N-ethyl-N′-dimethylaminopropylcarbodiimide (EDC) chemistry. This increase in signal resulted in a concomitant increase in sensitivity of detection, which facilitates analysis of low levels of A33 antigen. The apparent association rate (ka) and dissociation rate (kd) constants obtained with the different immobilisation chemistries were determined. These analyses showed that the kinetic constants obtained for the IgG were not significantly affected by the method of immobilisation. F(ab)2′ and Fab′ fragments immobilised using NHS/EDC chemistry showed significantly lower apparent affinity. By contrast the use of the thiol coupling chemistry with the Fab′ fragment gave a five fold increase in observed KA, resulting in a similar affinity to that observed with the intact IgG molecule.

Calcium channel block by (-)devapamil is affected by the sequence environment and composition of the phenylalkylamine receptor site

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.

Elucidation of the mechanism enhancing the avidity of lectin with oligosaccharides on the solid phase surface.

The mechanism underlying molecular recognition of lectins was elucidated by a novel solid phase binding assay system based on surface plasmon resonance. When the apparent affinities of interactions between chitooligosaccharides and wheat germ agglutinin were compared between lectin-immobilized and oligosaccharide-immobilized assay systems, the affinity constants (Ka) calculated for the former system were in good agreement with the previously reported values measured in solution. On the other hand, in the latter system, the calculated Ka could be more than 10,000 times higher than the values in solution at lower lectin concentrations. To elucidate the reason for this, we systematically investigated the effects of the oligosaccharide immobilized density and the lectin valence on the apparent affinity in the oligosaccharide-immobilized assay system. Both the apparent association (k[ass]) and dissociation rate constants (k[diss]) showed a tendency to decrease as the oligosaccharide density increased. This effect was most remarkable for the interaction possessing an extremely fast intrinsic k(ass). Oligomerization of lectin enhanced the avidity due to a significant reduction in k(diss). These phenomena could be explained by considering the nonhomogeneous conditions under which binding occurred. The reaction in a nonhomogeneous state is limited by the mass transport effect, and the effect of rebinding becomes so large that it cannot be disregarded. These findings are the first to demonstrate the importance of the mass transport effect in modulating the affinity of lectin for oligosaccharides on a solid phase surface.

Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses.

Although antibody affinity is primarily determined by immunoglobulin variable region structure human IgG antibodies of the four subclasses specific for the same antigen have been shown to differ in their affinity. To explore the influence of the immunoglobulin constant region on functional antibody affinity, a set of V region identical mouse-human chimeric IgG subclasses specific for TAG72 (tumour-associated glycoprotein) were studied. Biomolecular interaction analysis (BIA) was used to determine the binding kinetics of whole IgG subclasses and F(ab')2 fragments. Despite identical V regions, binding kinetics differed for the four subclasses. The apparent dissociation rate constants of the intact immunoglobulins ranked IgG4 < IgG3 < IgG2 < IgG1. In contrast, analysis of the binding characteriztics of the F(ab')2 fragments derived from IgG1, IgG2 and IgG4 revealed identical binding kinetics. The structure of the constant regions of the humanized IgG subclass antibodies clearly influenced functional antibody affinity, as has been described for the murine IgG subclasses. The exact mechanism for this phenomenon remains obscure but such differences should be taken into account when designing or choosing antibodies for therapeutic use.

The N-terminal B-domain of T4 gene 32 protein modulates the lifetime of cooperatively bound Gp32-ss nucleic acid complexes.

The N-terminal basic or B-domain (residues 1-21) of bacteriophage T4 gene 32 protein (gp32) provides a major determinant for highly cooperative binding by gp32 to single-stranded (ss) nucleic acids at equilibrium. In order to gain mechanistic insight into N-terminal domain function, the kinetics of dissociation of wild-type and previously characterized B-domain substitution mutant gp32s (R4K, R4Q, and K3A) from the model ribohomopolymer, poly(A), have been investigated under solution conditions identical to those used for equilibrium studies [Villemain, J. L., & Giedroc, D. P. (1993) Biochemistry 32, 11235-11246; Villemain, J. L., & Giedroc, D. P. (1996) J. Biol. Chem. 271, 27623-27629]. The dissociation of cooperatively bound gp32-poly(A) complexes was induced by sodium chloride concentration jumps and monitored by an increase in tryptophan fluorescence upon dissociation of the protein from poly(A) using stopped-flow techniques. The apparent dissociation rate constant, kd(app), for all mutant proteins studied was found to depend strongly on the initial fractional saturation of poly(A) just as was found previously for wild-type gp32. This permitted application of Lohman's model for the irreversible dissociation of cooperatively bound gp32-nucleic acid complexes [Lohman, T. M. (1983) Biopolymers 22, 1697-1713] from which the molecular rate constant, ke, the rate of dissociation of a protein monomer from teh end of a gp32-ss nucleic acid complex or protein cluster, could be determined. From the [NaCl]-dependence of kd(app), ke determined at 0.45 M NaCl, pH 8.1, 20 degrees C, was found to be 62 +/- 23, 78 +/- 8, 328 +/- 36, and 384 +/- 34 s-1 for wild-type, R4K, K3A, and R4Q gp32s, respectively. With the exception of R4K gp32, we find a striking correlation between the relative magnitudes of ke and Kapp, suggesting that the molecular defect in the equilibrium binding properties of the N-terminal domain mutants resides in the increased rate at which gp32 monomers dissociate from singly contiguous binding sites at the ends of clusters. The bimolecular association rate constant measured for wild-type gp32 and a weakly binding B-domain mutant, R4T gp32, to poly(dT) was found to be nearly identical, further evidence that the primary defect is in the dissociation reaction. We conclude that the N-terminal domain strongly modulates the lifetime of cooperatively bound gp32-polynucleotide complexes. The mechanistic and functional implications of these findings are discussed.

Specific micellar catalytic effects of the protolysis of ortho-Benzoic acid derivatives studied by the ultrasonic absorption method

Ultrasonic relaxation absorption has been observed in aqueous solutions of ortho-benzoic acid derivatives (OBAD) in the presence of a micelle of tetradecyltrimethylammonium bromide (TTAB). Relaxation absorptions were ascribed to the protolysis of acids on the surface of the micelle. The apparent dissociation constant (pKa=kb/γ2kf), rate constants (γ2kf, kb) and volume change (ΔV) for the reactions were determined. The dependence of rate constants on pKa for OBAD was compared with that for meta- and para-benzoic acid derivatives (BAD). It was especially noticeable that the dissociation of OBAD in the micellar solution is depressed more than that in aqueous solution. This can be explained as due to electrostatic effects, the polarity of solution and solvation in the TTAB micellar solution. The dependence of ΔV of OBAD on pKa was also compared with that of BAD. The result was considered to be due to differences in solute–solvent interactions and the situation around OBAD and BAD in the micellar solution.

Lophotoxin is a slow binding irreversible inhibitor of nicotinic acetylcholine receptors.

Lophotoxin and the bipinnatins are members of the lophotoxin family of marine neurotoxins, which covalently react with Tyr190 in the alpha-subunits of the nicotinic acetylcholine receptor. Bipinnatin-A, -B, and -C are protoxins that have been shown to spontaneously convert from inactive to active toxins during preincubation in buffer. However, in this report, we show that preincubation of lophotoxin did not result in an increase in the subsequent rate of irreversible inhibition of nicotinic receptors. Thus, unlike the bipinnatins, lophotoxin does not appear to be an inactive protoxin. Lophotoxin preferentially inhibited one of the two acetylcholine-binding sites on the receptor, and this preference resulted from both a higher reversible affinity and a faster rate of irreversible inhibition at this site. Association of 125I-alpha-bungarotoxin in the presence of lophotoxin was analyzed to obtain the apparent reversible association and dissociation rate constants for lophotoxin. The apparent association rate constant of lophotoxin was approximately 10(6)-fold slower than expected for a diffusion-limited interaction, indicating that lophotoxin is a slow binding irreversible inhibitor. The kinetic constants that describe the interaction of lophotoxin with the receptor did not change in the presence of dibucaine, suggesting that, unlike agonists, the slow apparent association of lophotoxin does not result from a slow transition of the receptor to a desensitized conformation.

Effect of antibody density on the displacement kinetics of a flow immunoassay

This study investigates the effect of antibody density on the kinetics of a solid-phase displacement immunoassay. Conducted in flow under nonequilibrium conditions, the assay utilizes a monoclonal antibody to the cocaine metabolite benzoylecgonine, which has been immobilized onto Sepharose beads and saturated with fluorophorelabeled antigen. Displacement of antibody-bound labeled antigen by non-labeled antigen occurs when sample is introduced in the buffer flow. Comparison of matrices coated with two different antibody densities revealed that the displacement efficiency is a function of the density of antibody-bound labeled antigen. A higher density of antibody provides a higher amount of displaced labeled antigen, but the displacement efficiency of the assay is decreased. The effect of antibody density on the immunoassay kinetics was analyzed using a mathematical formulation developed to characterize antibody-antigen interactions at solid-liquid interfaces. Higher antibody density proved to be associated with a lower apparent dissociation rate constant. The implications of these results on the design of immunassays in flow are discussed.

Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology.

The relative binding affinities of influenza virus N9 sialidase from term and whale with the Fab fragment of monoclonal antibody NC41 were determined using biosensor technology (Pharmacia BIAcoreTM). The apparent association and dissociation rate constants were measured in real time for the interaction of the Fab with both sialidases, the Fab being immobilised on the sensor surface. Although three-dimensional structural studies have shown that there are no apparent structural differences between the term and whale N9 sialidase epitopes to which the NC41 Fab binds, the apparent binding constant for the interaction with tern N9 sialidase was approximately 2.4-fold higher than that with whale N9 sialidase. The kinetic analysis showed that the association rate constant for the binding of whale N9 sialidase was higher than that for tern N9 sialidase (12.0 x 10(4) M-1 s-1 compared to 4.3 x 10(4) M-1 s-1) and the dissociation rate constants for the whale N9-sialidase-Fab complex were approximately 6-fold higher than for the tern N9-sialidase-Fab complex. Furthermore, kinetic analysis of the dissociation reaction showed that it was composed of two stages, an initial, faster rate followed by a late, slower rate. The values of the relative affinity constants calculated using the initial dissociation rate constant were similar to the values measured at equilibrium in the BIAcore and those determined in true solution equilibrium studies using sedimentation equilibrium. The late, slower, dissociation rate constant yielded affinity constants significantly higher than those obtained by true solution methods.

Antigen-Antibody Binding and Mass Transport by Convection and Diffusion to a Surface: A Two-Dimensional Computer Model of Binding and Dissociation Kinetics

The kinetics of binding and dissociation between a soluble analyte and an immobilized ligand on or near a surface are described numerically by an iterative computer model. The model is applied to a microflow chamber which is used for surface plasmon resonance measurements. It calculates diffusion perpendicular to the surface, flow parallel to the surface, and the interaction between any number of soluble and immobilized species. If the reaction between analyte and ligand is fast, binding and dissociation are influenced by the transport of the analyte to or away from the surface, in this case the measurement yields apparent association and dissociation rate constants which are not identical with the reaction rate of analyte and ligand. The transition between mass transport-controlled processes and reaction-controlled processes is described and attention is drawn to possible misinterpretations of experimental binding and dissociation curves. The measurement of rate constants higher than allowed by the conventional technique can be performed by elution of the analyte with a second analyte of low molecular weight.The kinetics of binding and dissociation between a soluble analyte and an immobilized ligand on or near a surface are described numerically by an iterative computer model. The model is applied to a microflow chamber which is used for surface plasmon resonance measurements. It calculates diffusion perpendicular to the surface, flow parallel to the surface, and the interaction between any number of soluble and immobilized species. If the reaction between analyte and ligand is fast, binding and dissociation are influenced by the transport of the analyte to or away from the surface, in this case the measurement yields apparent association and dissociation rate constants which are not identical with the reaction rate of analyte and ligand. The transition between mass transport-controlled processes and reaction-controlled processes is described and attention is drawn to possible misinterpretations of experimental binding and dissociation curves. The measurement of rate constants higher than allowed by the conventional technique can be performed by elution of the analyte with a second analyte of low molecular weight.

Evidence of an associative intermediate on the myoglobin refolding pathway

Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin. This is the only method to date that yields direct information on protein physical dimensions during the folding process. It has the potential to detect and probe important processes, such as protein compaction and association, on a millisecond time scale. Initial experiments were performed with horse myoglobin denatured in high concentrations of urea. The denatured protein was diluted rapidly into a buffer containing no urea or low concentrations of urea. The time-course of the forward-scattered intensity shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process. Initial and final radii of gyration correspond well to a dimer and a monomer, respectively. Kratky plots of the initial and final states also support the transient dimerization model. The apparent dissociation rate constant was obtainable directly from the data. An association rate constant and an equilibrium constant could be estimated. The dimerizing intermediate is speculated to be a globular non-native state with an exposed hydrophobic surface.

Experimental and theoretical studies of rate constant evaluation by affinity chromatography Determination of rate constants for the interaction of saccharides with concanavalin A

Experimental studies have been combined with numerical simulations of frontal affinity chromatography to illustrate the potentials of this technique for obtaining rate constants for the interaction of the solute with affinity matrix and for a reaction in competition with the solute—matrix interaction. Frontal chromatography of p-nitrophenylmannoside on a standard HPLC column of concanavalin A—CPG 3000 at flow-rates of 2–10 ml/min has yielded a rate constant of 0.42 s −1 for the dissociation of the solute-immobilized lectin complex on the basis of the flow-rate dependence of boundary spreading in the elution profile: this constant ( k −1) has been obtained by extrapolating the apparent dissociation rate constant ( k obs −1) to zero solute concentration to eliminate the consequences of non-linear (Langmuir) kinetics. Numerical simulations of the affinity chromatographic behaviour of p-nitrophenylmannside (A) in the presence of a fixed concentration of a second saccharide (B) that competes for matrix sites have shown that the corresponding extrapolation of k obs −1 to zero solute concentration again yields k −1, but that the presence of competing saccharide is manifested as a change in slope of the essentially linear dependence of k obs −1 upon p-nitrophenylmannoside concentration ([A]). A calibration plot of the variation of d( k obs −1/d[A] with dissociation rate constant for the competing ligand—matrix interaction ( k −2) has been used to obtain a rate constant of 1 s −1 for the dissociation of methylmannoside from the immobilized lectin on the basis frontal affinity chromatography of p-nitrophenylmannoside in the presence of 100μ M competing saccharide. Finally, the potential of affinity chromatography for the evaluating the dissociation rate constant for a competing interaction between ligand and solute has been illustrated by numerical simulation of elution profiles and their interpretation in terms of a published expression for the flow-rate dependence of boundary spreading under conditions of linear kinetics; and by extrapolation of the rate constants so obtained to zero solute concentration to take into account the effects of Langmuir kinetics on chromatographic migration.

Amino acid substitutions at position 47 of human beta 1 beta 1 and beta 2 beta 2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants.

Human liver alcohol dehydrogenase isoenzymes beta 1 beta 1 and beta 2 beta 2, in which position 47 in the coenzyme binding domain is an arginine or histidine, respectively, differ remarkably in steady-state kinetics. To understand which catalytic steps affect these kinetics, apparent coenzyme dissociation and association rate constants, and apparent 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) hydride transfer rate constants were obtained with stopped-flow kinetics. Enzymes containing site-specific mutations of Arg-47 in beta 1 beta 1 (beta 47R) to His (beta 2 beta 2 or beta 47H), Lys (beta 47K), or Gln (beta 47Q) were studied. Apparent coenzyme dissociation rate constants are greatly affected by substitutions at position 47, in which mutant enzymes with a weak base or a neutral residue at this position (beta 47H and beta 47Q) exhibit faster rate constants than beta 47R and beta 47K. Substitutions at position 47 have less effect on apparent coenzyme association rate constants. The kinetics of NADH association for beta 47H and beta 47Q are consistent with a two-step mechanism in which the bimolecular binding step is coupled to a unimolecular process. These findings indicate that the greater role of position 47 in coenzyme dissociation may occur after a coenzyme-induced isomerization. Substitutions at position 47 also strongly influence apparent DACA hydride transfer rate constants; hydride transfer is faster with mutant enzymes containing weak bases like histidine at this position. Steady-state kinetics, however, reveal that the rate-limiting step of both beta 47R and beta 47H for acetaldehyde reduction and for ethanol oxidation is coenzyme product dissociation. Thus, the different activities of beta 1 beta 1 and beta 2 beta 2 for ethanol oxidation and acetaldehyde reduction are caused primarily by different coenzyme dissociation rates.

Kinetics of antibody binding at solid-liquid interfaces in flow

We have developed the theoretical framework for a displacement immunoassay conducted in flow under nonequilibrium conditions. Using a repetitive displacement technique, we determined the displacement rate and apparent dissociation rate constant at different flow rates. Our data suggest that the kinetics are best described by a first-order function. The displacement efficiency, the displacement rate, and therefore the apparent dissociation rate constant were calculated and demonstrated to be flow rate dependent. The theoretical framework developed in this study was successful in predicting the behavior of antigen displacement in flow.